Bone resorption by osteoclasts (OCs) exceeding formation by osteoblasts (OBs) is a biological problem with a central role in widespread and costly disorders, including osteoporosis, osteo- and rheumatoid arthritis, periodontal disease, and prosthesis/implant loosening. Delineation of mechanisms and regulation of the fusion of mononuclear precursors into multinucleated OCs is important since fusion is needed for normal bone resorption. In our studies of gene expression changes during OC differentiation, we discovered OC-STAMP (osteoclast-stimulatory transmembrane protein) and noted a series of striking similarities to a factor shown by others to be essential to fusion, called DC-STAMP. Similarities include 1) up- regulation of mRNA and protein upon stimulation of OC precursors by RANKL; 2) predicted topology of transmembrane (TM) helices; 3) presence of a DC-STAMP family consensus sequence in the C-terminal half of OC-STAMP; 4) suppression of formation of multinucleated osteoclasts in vivo and in vitro by either knockout, knockdown, or antibody; 5) stimulation of fusion upon overexpression. Both proteins are highly conserved in terrestrial vertebrates. In a model of acute, induced OC differentiation in an osteopetrotic rat model, we present preliminary evidence that anti-OC-STAMP antibody suppresses osteoclastogenesis in vivo. [A recent report validated our hypothesis that OC-STAMP -/- mice would, like DC-STAMP -/- mice, have mononuclear OCs.] This proposal will investigate pre-OC fusion under 3 Specific Aims to clarify its role in OC cell biology and activity in vivo, as a potential therapeutic targetto control bone loss, and in relation to DC- STAMP as a fusion factor. SA1 will establish the membrane topology of both OC- and DC-STAMP (DC- STAMP has been reported to be a 7-TM superfamily member, yet different algorithms predict different topologies). Information gained should facilitate next-generation antibodies and/or inhibitory peptides directed at proven extracellular loops of these fusigens and provide a basis for further hypotheses about activities and regulation. Under SA2, OC- and DC-STAMP knockout mice will be produced (requests for existing lines were refused). We will confirm the skeletal and OC phenotypes of both strains in our hands, and we will use cells derived from them to perform fusion experiments to determine interaction or interdependence of OC- and DC- STAMP for fusion. Studies will be done to identify potential ligands and regulatory factors for OC- and DC- STAMP. Under SA3, we will increase the size of our preliminary study of acute, in vivo OC inhibition in tl/tl rats with anti-C-STAMP antibody. We will also investigate the effects of anti-[fusigens] antibody inhibition in the proven, ovariectomized rat model of osteoporosis using athymic rats. Together, these studies should provide significant new information about the topology and cell biology of both OC-STAMP and DC-STAMP, about pre- OC fusion, about the phenotypic impact of loss of OC-STAMP on the skeleton, and whether in principle, inhibition of pre-OC fusion is a valid approach to stemming bone loss in vivo.